Conjugate between a modified superantigen and a target-seeking compound and the use of the conjugate

ABSTRACT

Methods of lysing cells associated with a disease condition, and method of treating a disease condition by lysing cells associated with the condition, by administering to a mammal a therapeutically effective amount of a conjugate comprising a biospecific affinity counterpart and a peptide that is derived from Staphylococcal enterotoxin A, that has the ability to bind to a Vβ of a T cell receptor and has been modified at amino acid position 47, 128, 187, 225 or 227, in order to have reduced ability to bind to MHC class II antigens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. patent application Ser. No.08/765,695, filed Jul. 25, 1997, which is a U.S. national phase Section371 application of PCT Patent Application Number PCT/SE95/00681, filedJun. 7, 1995, which claims priority to Swedish patent application number9402430-4 filed Jul. 11, 1994, each of which are incorporated herein byreference in their entireties, and all priorities are claimed.

TECHNICAL FIELD BACKGROUND OF THE INVENTION

Superantigens are primarily proteins of viral or bacterial origin andare capable of simultaneous binding to MHC class II antigens onmammalian cells and the T cell receptor Vβ chain. The binding leads toactivation of T-lymphocytes and lysis of the MHC class II bearing cells.The moderate degree of polymorphism of the binding part of the Vβ chaincauses a relatively large portion of the T-lymphocytes to be activatedwhen contacted with a superantigen (in comparison with activationthrough normal antigen-processing).

Initially the superantigen concept was associated with variousstaphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SED, and SEE).Recently a new staphylococcal enterotoxin named SEH has been discovered(Keyong et al., J. Exp. Med. 180 (1994) 1675-1683). After the interesthad been raised, further superantigens were discovered. Examples areToxic Shock Syndrome Toxin 1 (TSST-1), Exfoliating Toxins (Exft) thatare associated with scalded skin syndrome, Streptococcal PyrogenicExotoxin A, B and C(SPE A, B, and C), Mouse Mammary Tumor Virus Proteins(MMTV), Streptococcal, M Proteins, Clostridial perfringens enterotoxin(CPET) among others. For a review of superantigens and their propertiessee Kotzin et al. (Adv. Immunol. 54 (1993) 99-166).

Pseudomonas exotoxin A has been looked upon as a functional superantigenbecause there are results indicating that this toxin may be processedintracellularly by accessory cells to fragments that are expressed onthe cell surface with the ability to bind to the Vβ chain and asubsequent activation of T cells. (Pseudomonas exotoxin A. Legaard etal., Cell. Immunol. 135 (1991) 372-382).

Superantigens as such have been suggested for therapy of variousdiseases with curative effects being accomplished through a generalactivation of the immune system (Kalland et al., WO 9104053; Terman etal., WO 9110680; Terman et al., WO 9324136; Newell et al., Proc. Natl.Acad. Sci. USA 88 (1991) 1074-1078).

In connection with vaccines it has been suggested to use superantigensthat have been mutated so as to lose their TCR binding ability (Kappler& Marrack, WO 9314634).

The mutation of superantigens has previously been described (Kappler &Marrack, WO 9314634; Kappler et al., J. Exp. Med. 175 (1992) 387-396;Grossman et al., J. Immunol. 147 (1991) 32743281; Huffnagle et al.,Infect. Immun. 59 (1991) 2126-2134).

We ourselves have previously suggested to employ conjugates between asuperantigen and an antibody for therapy in order to lyse cells thatexpress the structure towards which the antibody is directed (Dohlstenet al., WO 9201470; Lando et al., Cancer Immunol. Immunother. 36 (1993)223-228; Kalland et al., Med. Oncol. Tumor Pharmacother. 10 (1993)37-47; Lando et al., J. Immunol. 150 (8 part 2) (1993) 114A (JointMeeting of the American Association of Immunologists and the ClinicalImmunology Society, Denver, Colo., USA, May 21-25 (1993)); Lando et al.,Proc. Am. Assoc. Cancer Res. Annu. Meet. 33(0) (1992) 339 (Annualmeeting of the American Association for Cancer Research, San Diego,Calif., USA, May 20-23 (1992)); Dohlsten et al., Proc. Natl. Acad. Sci.USA 88 (1991) 9287-9291). Diseases suggested to be treated have beencancers, viral infections, parasitic infestations, autoimmune diseasesand other diseases associated with cells expressing disease-specificsurface structures. The experimental work carried out so far has focusedon conjugates containing recombinant SEA and various anti-cancerantibodies. The conjugates as such have had a somewhat reduced abilityto bind MHC class II antigens compared to the non-conjugated form of thesuperantigen. It has not been determined if a decreased MHC class IIantigen binding ability is beneficial or not for achieving an optimallyse and an optimal therapeutic effect.

Immune therapy experiments with SEB chemically conjugated to a tumorspecific anti-idiotype antibody have previously been described by Ochiet al., (J. Immunol. 151 (1993) 3180-3186).

During the prosecution of the priority application the Swedish PatentOffice has additionally cited Buelow et al. (J. Immunol. 148 (1992) 1-6)that describes fusions between Protein A and fragments of SEB withoutemphasis of the MHC classs II binding or use of the fusion for cellkilling; and Hartwig et al. (Int. Immunol. 5 (1993) 869-875) thatdescribes mutations affecting MHC class II binding of the non-fused formof the superantigen streptococcal erythrogenic toxin A.

BRIEF SUMMARY OF THE INVENTION

A first objective of the invention is to improve previously knownsuperantigen-antibody conjugates with respect to general immunestimulation versus directed cytotoxicity. Stimulation results inactivated T-lymphocytes and is dependent on the ability of thesuperantigen to bind to both the T cell receptor and an MHC class IIantigen.

A second objective of the invention is to provide conjugates betweenbiospecific affinity counterparts (e.g. antibodies) and superantigenswith a modified affinity for NHC class II antigens. This has now beenshown to improve the selectivity for superantigen antibody dependentcell cytolysis (SADCC) of cells exposing the antigen (against which theantibody/biospecific affinity counterpart of the conjugate is directed)over other cells exposing MHC class II antigens.

A third objective of the invention is to provide conjugates that can beused as the active principle in the treatment of mammals suffering fromcancers, autoimmune diseases, parasitic infestations, viral infectionsor other diseases associated with cells that on their surface expressstructures that are specific for respective disease.

BRIEF DESCRIPTION OF THE DRAWINGS

General

The mutant SEA(D227A) (=SEA(m9) or mutant m9) was at the priority datethe most promising SEA variant. We have therefore selected to present invitro and in vivo results with this variant (FIGS. 3-6).

FIG. 1 is a schematic outline of the plasmids used to express SEA andC215Fab-SEA. The coding regions and the two transcription terminatorsfollowing the product genes are indicated by boxes. The gene encodingthe kanamycin resistance protein is labeled Km. lacI is the lacrepressor gene. V_(H) and C_(H)1 indicates the gene encoding the F_(d)fragment of the heavy chain of the murine antibody C215. Likewise V_(K)and C_(K) indicates the gene encoding the kappa chain. Rop is the geneencoding the replication control protein from pBR322. The promotersdirecting transcription of product genes are shown as arrows, in pKP889the trc promoter and in the other two vectors the promoter fromstaphylococcal protein A (spa). The region containing the origin ofreplication is indicated by ori. The only difference between SEA encodedby pKP943 and pKP1055 is a glycine residue added at the N-terminus ofthe latter. The SEA gene contained in the latter vector also containsmore unique restriction enzyme sites, introduced by silent mutations.

FIG. 2 is a circular dichroism spectra for wild-type SEA and for themutants F47A and D227A, representing the most severely reduced mutationsin each MHC class II binding region. The solid line is the curve forwild-type SEA. The curves for the mutants are dotted or center, F47Arespectively D227A.

FIG. 3 shows the concentration dependency of superantigen dependentmediated cellular cytotoxicity (SDCC) for SEA(wt) and SEA(D227A).

FIG. 4 shows the concentration dependency of superantigen dependent cellmediated cytotoxicity (SDCC) for C215Fab-SEA(wt) and C215Fab-SEA(D227A).

FIG. 5 shows the concentration dependency of superantigen mAb dependentcell mediated cytotoxicity (SADCC) for C215Fab-SEA(wt) andC215Fab-SEA(D227A) compared to free SEA(wt).

FIG. 6A compares the therapeutic effects obtained in C57B1/6 micecarrying lung metastases of B16-C215 melanoma cells by treatment withC215Fab-SEA(wt) and C215Fab-SEA(D227A).

FIG. 6B shows toxicity of C215-SEA(wt) and C215-SEA(D227A) for thetreatments represented in FIG. 6 a.

DETAILED DESCRIPTION OF THE INVENTION

The main aspect of the invention is a conjugate comprising:

-   -   a. a biospecific affinity counterpart that is directed towards a        structure to which one intends to bind to the conjugate,    -   b. a peptide that        -   i. is derived from a superantigen,        -   ii. has the ability to bind to the Vβ chain of the T cell            receptor, and        -   iii. has a modified ability to bind to MHC class II antigens            compared to the superantigen from which the peptide is            derived (wild-type of superantigen=SA(wt)).

The peptide and the affinity counterpart are covalently linked to eachother via a bridge (B).

The preferred conjugates have the ability to activate and directT-lymphocytes to selective lysis of cells that on their surface exposethe structure against which the affinity counterpart is directed. Thismeans that the conjugates shall cause cytolysis in an SADCC mediatedmethod (Superantigen Antibody Dependent Cellular Cytotoxicity). See theexperimental part below and our previous publications concerningconjugates between superantigens and antibodies (e.g. Dohlsten et al.,WO 9201470).

The inventive conjugates have a structure that is analogous to thesuperantigen-antibody conjugates described in the prior art (Dohlsten etal., WO 9201470 which hereby is incorporated by reference), i.e. theconjugates complies with the formula:T-B-SA(M)where T represents the biospecific affinity counterpart, SA(m) is themodified superantigen (the above-mentioned peptide), and B is a covalentbridge linking T and SA(m) together.

T can in principle be any structure that binds via biospecific affinity.In most important cases, T is capable of binding to a cell surfacestructure, preferably a disease specific structure as given above. Thestructure against which T is directed is usually different from (a) theVβ chain epitope to which the superantigen derived peptide (SA(m)) bindsand (b) the MHC class II antigen epitope to which the unmodifiedsuperantigen binds. The biospecific affinity counterpart T may primarilybe selected among interleukins (e.g. interleukin-2), hormones,antibodies and antigen binding fragments of antibodies, growth factorsetc. See for instance Woodworth, Preclinical and Clinical Development ofCytokine Toxins presented at the conference “Molecular Approaches tocancer Immunotherapy”, Ashville, N.C., Nov. 7-11, 1993. Polypeptidesbinding to the constant domains of immunoglobulins (e.g. Proteins A andG and L), lectins, streptavidin, biotin etc were at the priority dateconsidered to be of minor importance.

At the priority date, it was preferred that T was an antibody or anantigen binding fragment of an antibody (including Fab, F(ab)₂, Fv,single chain antibody etc), with particular emphasis of an antibodyactive fragment (such as Fab) of antibodies directed against the socalled C242 epitope (Lindholm et al., WO 9301303) or against othercancer specific epitopes.

In case T is an antibody it is primarily monoclonal or a mixture of adefined number of monoclonals (e.g. 2, 3, 4, 5 or more). T may be apolyclonal antibody, in case the use is nontherapeutical.

It is not imperative for T to have a polypeptide structure.

The modified superantigen SA(m) is primarily a mutated superantigen butmay potentially also be a chemically modified superantigen, includingfragments of superantigens retaining the ability to bind to the Vβ chainof the T cell receptor.

The expression “mutated superantigen” means that the native ability ofthe superantigen to bind to MHC class II antigens has been modified onthe genomic level by replacing, inserting or removing one or more aminoacids in the native superantigen.

Superantigen fragments obtained by mutations removing parts of the fullamino acid sequence and fragments obtained by enzymatic or chemicalcleavage of superantigens may be used equivalently in chemicalconjugates of the invention.

The modified superantigen SA(m) may comprise one or more amino acidsequences that are derived from different superantigens and that mayhave been mutated, for instance combinations of the preferredsuperantigens mentioned below.

The modified superantigen SA(m) as such may exhibit a decreasedimmunogenicity and toxicity compared to the native superantigen.

Other groups/substances that are capable of cross reacting with the VPchain of the T cell receptor may potentially also be employedequivalently with the mutated superantigen (SA(m)) as given above. Suchgroups/substances may be of non-polypeptide structure.

At the end of the priority year the most interesting product candidatesof the invention comprised mutated forms of superantigens havingmultiple MHC class II binding sites and/or the ability to coordinateZn²⁺, for instance SEA, SED, SEE and SEH.

T as well as SA(m) may be prepared by recombinant techniques.

The bridge B may be selected as previously described (Dohlsten et al.,WO 9201470), i.e. it shall preferably be hydrophilic and exhibit one ormore structure(s) selected among amide, thioether, ether, disulfide etc.In case the bridge have unsubstituted unbroken hydrocarbon chains theypreferably lack aromatic rings, such as phenyl. The most importantbridges are those obtained by recombinant techniques, i.e. when theconjugation takes places on the genomic level. In such casesoligopeptide bridges containing hydrophilic amino acid residues, such asGln, Ser, Gly, Glu and Arg, are preferred. Pro and His may also beincluded. During the priority year it has been decided that thepreferred bridge is a peptide comprising three amino acid residues(GlyGlyPro).

The inventive conjugate may comprise one or more modifiedsuperantigen(s) per biospecific affinity counterpart and vice versa.This means that T in the formula above may contain one or more modifiedsuper antigens in addition to the biospecific counterpart. In analogySA(m) may contain one or more biospecific affinity counterpart(s) T. Theaffinity counterpart T and SA(m) may also comprise other structures. Thenumber of modified superantigens per affinity counterpart is preferablyone or two.

The synthesis of the novel inventive conjugates may be carried out inprinciple according to two main routes: 1. by recombinant techniques and2. chemical linking of T to SA(m). The methods are well recognized forthe ordinary skilled worker in the field and comprise a large number ofvariants. It follows that the invention primarily concerns artificialconjugates, i.e. conjugates that are not found in nature.

Chemical linking of a modified superantigen to the biospecific affinitycounterpart T often utilizes functional groups (e.g. primary aminogroups or carboxy groups) that are present at many positions in eachcompound. It follows that the final product will contain a mixture ofconjugate molecules differing with respect to the position at whichlinking has taken place.

For recombinant conjugates (fusion proteins) the obtained conjugatesubstance will be uniform with respect to the linking position. Eitherthe amino terminal of the modified superantigen is linked to the carboxyterminal of the biospecific affinity counterpart or vice versa. Forantibodies, such as intact antibodies and antigen binding fragments(Fab, Fv etc), either the light or the heavy chain may be utilized forsuch fusions. At present time recombinant conjugates are preferred, withpreference for Fab fragments and linking of the amino terminal of themodified superantigen to the first constant domain of the heavy antibodychain(CH1), without exclusion of the analogous linking to the lightchain or to the VH and VL domain that also may give quite good results.

There are two different methods for obtaining large amounts ofsuperantigens (including modified and fused forms) in E. coli:intracellular production or secretion. The latter method is preferredfor the inventive conjugates because it offers purification of correctlyfolded protein from the periplasma and from the culture medium.Intracellular production results in a complicated purification procedureand often needs refolding in vitro of the protein (in order for theprotein to obtain the correct tertiary structure). The above does notexclude that it is possible to produce active conjugates also in otherhost cells, e.g. eukaryotic cells, such as yeast or mammalian cells.

The production of mutated superantigens and selection of mutants havinga modified ability to bind (affinity) to MHC class II antigens maybecarried out according to known techniques (See e.g. Kappler et al., J.Exp. Med. 165 (1992) 387-396). See also our experimental part.

The ability of the conjugate to bind to the T cell receptor Vβ chain, tothe target structure and to cause lysis of the target cell depends oni.a. the peptide (SA(m)) that is derived from a superantigen, thebiospecific affinity counterpart (T) and the structure and length of thebridge (B). A person ordinary skilled in the art is able to optimize theinventive conjugates with respect to the binding ability and the abilityto cause lysis by studying the relationship between effect and structurewith the aid of those models that have been disclosed in connection withpreviously known superantigen antibody conjugates (see theabove-referred publications). See also the experimental part below. Theability of the conjugate to bind to the T cell receptor Vβ chain, to thetarget structure and to cause lysis of the target cell depends on i.a.the peptide (SA(m)) that is derived from a superantigen, the biospecificaffinity counterpart (T) and the structure and length of the bridge (B).A person ordinary skilled in the art is able to optimize the inventiveconjugates with respect to the binding ability and the ability to causelysis by studying the relationship between effect and structure with theaid of those models that have been disclosed in connection withpreviously known superantigen antibody conjugates (see theabove-referred publications). See also the experimental part below.

By modified ability to bind MHC class II antigens is primarily intendedthat the ratio IC₅₀(SA(wt)):IC₅₀(SA(m)) is <0.9 (90%), such as <0.5(<50%) and possibly also <0.01 (<1%). In the alternative the modifiedbinding ability of the inventive conjugates can be measured as the ratioof the dissociation constants K_(d)(SA(wt)):K_(d)(SA(m)) with K_(d)measured in nM and with the same limits as for the ratioIC₅₀(SA(wt)):IC₅₀(SA(m)). For the determination of IC₅₀(SA(wt),IC₅₀(SA(m)), K_(d)(SA(m)) and K_(d)(SA(m)) see the experimental partbelow.

It is previously known that certain superantigens may have two or moresites that bind to MHC class II antigen (Fraser et al., In:Superantigens: A pathogens view on the immune system. Eds. Huber &Palmer, Current Communications in Cell Molecular Biology 7 (1993) 7-29).For this type of superantigens the binding ability shall be modified atleast one of the binding sites, e.g. as a reduction of theabove-mentioned size. Possibly it may suffice with a superantigenmodification that create a changed difference in affinity for two MHCclass II binding sites, tentatively >10% and preferably by reducing theaffinity of at least one site.

Superantigens bind to TCR Vβ chains of different subgroups with varyingaffinities. In the inventive fusion proteins/conjugates, thesuperantigen employed may have been modified so as to show an alteredsubgroup specificity or an altered affinity to one or more members ofthe subgroup. There are strong reasons to believe that a parabolicrelationship exists between the affinity for TCR Vβ and stimulation viaTCR, i.e. a moderate affinity will give the maximal stimulation.Accordingly an appropriate affinity of a modified superantigen for TCRVβ may be at hand as soon as the fusion protein/conjugate comprising themodified superantigen is able to significantly stimulate a resting Tcell population representing essentially the distribution of all humanVβ subgroups to proliferate. The T cell population may be pooled T cellsfrom randomly selected human individuals. By significantly is meant thatthe stimulation is possible to measure. The results presented in TableII (right column) in the experimental part indicate that the ability tocause SADCC of the inventive conjugates/fusion proteins often isessentially the same as for the fusion comprising the wild-typesuperantigen.

Main Use of the Conjugates/Fusion Proteins of the Invention

The conjugates according to the invention are primarily intended for thetreatment of the same diseases as the conjugates between normalsuperantigens and antibodies. See the abovementioned publications. Thusthe inventive conjugates may be administered either as the main therapyor as adjuvant therapy in connection with surgery or other drugs.

The pharmaceutical composition of the invention comprises formulationsthat as such are known within the field but now containing our novelconjugate. Thus the compositions may be in the form of a lyophilizedparticulate material, a sterile or aseptically produced solution, atablet, an ampoule etc. Vehicles such as water (preferably buffered to aphysiologically pH value by for instance PBS) or other inert solid orliquid material may be present. In general terms the compositions areprepared by the conjugate being mixed with, dissolved in, bound to, orotherwise combined with one or more water-soluble or water-insolubleaqueous or non-aqueous vehicles, if necessary together with suitableadditives and adjuvants. It is imperative that the vehicles andconditions shall not adversely affect the activity of the conjugate.Water as such is comprised within the expression vehicles.

Normally the conjugates will be sold and administered in predispenseddosages, each one containing an effective amount of the conjugate that,based on the result now presented, is believed to be within the range of10 μg-50 mg. The exact dosage varies from case to case and depends onthe patient's weight and age, administration route, type of disease,antibody, superantigen, linkage (—B—) et.

The administration routes are those commonly known within the field,i.e. a target cell lysing effective amount or a therapeuticallyeffective amount of a conjugate according to the invention is contactedwith the target cells. For the indications specified above this mostlymeans parenteral administration, such as injection or infusion(subcutaneously, intravenously, intra-arterial, intramuscularly) to amammal, such as a human being. The conjugate may be administered locallyor systemically.

By “target cell lysing effective amount” is contemplated that the amountis effective in activating and directing T-lymphocytes to destroy thetarget cell.

At the end of the priority year it had been decided that the preferredadministration route for conjugates/fusion proteins comprisingunmodified superantigens is 3 hours' intravenous infusion per daycombined with a fever-reducing agent (paracetamol). The administrationis to be repeated during 4 days and stopped before secondary antibodiesare raised against the fusion protein/conjugate in the patient. Thisdosage schedule is likely to be applicable also to the present inventiveconjugates/fusion proteins.

Alternative Fields of Use

The inventive conjugates can also be employed to quantitatively orqualitatively detect the structure against which the target-seekinggroup (T) is directed. In general these methods are well-known to peoplein the field. Thus, the modified superantigen may function as a markergroup within immunoassays including immunohistochemistry meaning thatthe marker group in turn is detected by for instance an antibody that isdirected towards the peptide (SA(m)) and labeled with an enzyme,isotope, fluorophor or some other marker group known per se. Anotherimmunoassay method is to detect in a cell population cells that on theirsurface express a structure capable of binding to the target-seekinggroup (T). This use means that a sample from the cell population isincubated with T-lymphocytes together with the present inventiveconjugate as in an SADCC assay. In case the incubation leads to celllysis this is an indication that the population contains cells that ontheir surface express the structure.

Experimental Part Manufacture of Recombinant Proteins

Antibodies

The experimental work in connection with the invention has primarilybeen done with monoclonal antibody C215 as a model substance. Thisantibody is directed against an antigen in the GA-733 family (see forinstance EP 376,746) and references cited therein and Larsson et al.,Int. J. Canc. 32 (1988) 877-82). The C215 epitope has been judged not tobe sufficiently specific for cancer treatment in humans. At the prioritydate mab C242 (Lindholm et al., WO 9301303) was believed to be a bettercandidate, as judged from experiments with its fusion product withwild-type SEA.

Bacterial Strains and Plasmids

The E. coli strains UL635 (xy1-7, ara-14, T4^(R), ΔompT) and HB101(Boyer and Roulland-Dessoix, J. Mol. Biol. 41 (1969) 459-472) were usedfor the expression and cloning, respectively. The vector pKP889 was usedfor expression of Fab-SEA fusion proteins (derived from the murineantibody C215) and the vectors pKP943 and pKP1055 for secretion of SEA(FIG. 1). The Fab-SEA expression vector pKP889 is identical to pKP865(Dohlsten et al, Proc. Natl. Acad. Sci. USA (1994) in press) except thatthe spacer between CH1 and SEA is GlyGlyAlaAlaHisTyrGly. Expression frompKP943 yields SEA with the native amino terminus. The use of pKP1055results in SEA having a Gly residue added at the amino terminus. In bothvectors the signals from staphylococcal protein A (Uhlén et al., J.Biol. Chem. 259 (1984) 1695-1702) are used for transcription andtranslation and a synthetic signal peptide for secretion (L. Abrahmsén,unpublished).

In Vitro Mutagenesis

Mutations were made by polymerase chain reactions run on a Perkin ElmerThermocycler. The reaction mixture (100 μl) contained: 1×PCR buffer fromPerkin Elmer Cetus (10 mM Tris/HCl pH 8.3, 1.5 mMMgCl₂, 0.001% (w/v)gelatine, an additional 2 mM MgCl₂, 0.4 mM dNTPs (Perkin Elmer Cetus),2.5 units of Ampli Taq DNA polymerase (Perkin Elmer Cetus, USA) and 100ng DNA template. Primers were added to a final concentration of 0.8 μM.The original template was a plasmid containing Staphylococcus aureusenterotoxin A gene identical to the one published by Betley et al. (J.Bacteriol. 170 (1988) 34-41), except that the first codon (encoding Ser)was changed to TCC to furnish a Bam HI site at the 5′ end of the gene.Later a derivative containing more unique restriction enzyme sitesintroduced by silent mutations was used. Mutations introduced next to arestriction site were made with one set of primers, one of thesespanning the mutation and the restriction site. For most mutations twoset of primers had to be used and the PCR was performed in twoconsecutive steps. A new restriction enzyme site was introduced togetherwith each mutation to enable facile identification. Oligonucleotidesused as primers were synthesized on a Gene Assembler (Pharmacia BiotechAB, Sweden). To confirm each mutation the relevant portion of thenucleotide sequence was determined on an Applied BiosystemsDNA-Sequenser using their Taq DyeDeoxy Termination Cycle Sequencing Kit.

Protein Production and Analysis

E. coli cells harboring the different gene constructs were grownovernight at room temperature (Fab-SEA vectors) and at 24-34° C.(secretion vectors, the optimum depends on the mutation). The broth was2×YT (16 g/l Bacto trypton, 10 g/l Bacto yeast extract, 5 g/l NaCl)supplemented with kanamycin (50 mg/l). Fusion proteins were induced byaddition of isopropyl-β-D-thiogalactoside to a final concentration of100 μM. (The protein A promotor used in the expression of non-fused SEAis constitutive). The cells were pelleted at 5000×. g and theperiplasmic contents were released by gently thawing the previouslyfrozen cell pellet in 10 mM Tris-HCl (pH 7.5) on ice during agitationfor 1 hour. The periplasmic extracts were clarified by centrifugation at9500×g for 15 minutes. The Fab-SEA proteins were used without furtherpurification. SEA and Gly-SEA were further purified by affinitychromatography on an anti-SEA antibody column. Polyclonal rabbitanti-SEA antibodies were previously collected from rabbits preimmunizedwith SEA and purified by affinity chromatography on protein GSepharoses® (Pharmacia Biotech).

Protein Analysis

The proteins were separated in precast polyacrylamide SDS Tris-GlycineNovex gels (gradient 4-20% or homogenous 12%, Novex novel experimentaltechnology) and either stained with Coomassie Blue or used in Westernblot. Polyclonal rabbit anti-SEA antibodies (above) were used to detectSEA in Western blot analysis, followed by porcine anti-rabbit Igantibodies, and rabbit anti-horseradish peroxidase antibodies andperoxidase. With Fab-SEA fusion proteins peroxidase conjugated ratantibodies recognizing the kappa chain were also used (AAC 08P, SerotechLTD, England). 3,3′-diaminobenzidine (Sigma) was used for visualizationof peroxidase.

Circular dichroism (CD) spectra were collected in a J-720spectropolarimeter (JASCO, Japan) at room temperature (22-25° C.) in 10mM phosphate buffer, pH 8.2, with 0.02 mM ZnSO₄ and 0.005% (v/v) Tween®20. The scanning speed was 10 nm/min and each spectrum was averaged fromfive subsequent scans. The cell path length was 1 mm and the proteinconcentration 0.2 to 0.5 mg/ml. Guanidine hydrochloride (Gdn-HCl)denaturations at equilibrium were measured at 23° C. by CD at 222 nmwith a protein concentration of 0.3 mg/ml and a cell path length of 1mm. These data were used to calculate the apparent fraction of unfoldedprotein (F_(app)). Equilibrium unfolding parameters were derived byfitting the data to a two-site folding process (Hurle et al.,Biochemistry 29 (1990) 4410-4419).

Binding and Functional Assays In Vitro

Materials

Reagents: RPMI 1640 medium obtained from Gibco, Middlesex, England wasused. The medium had a pH of 7.4 and contained 2 mM L-glutamine (Gibco,Middlesex, England), 0.01M HEPES (Biological Industries, Israel), 1 mMNaHCO₃ (Biochrom AG, Germany), 0.1 mg/ml Gentamycin sulphate (BiologicalIndustries, Israel), 1 mM Na-pyruvate (JRH Biosciences Industries, USA),0.05 mM mercaptoethanol (Sigma Co., USA), 100 times concentratednonessential amino acids (Flow Laboratories, Scotland) and wassupplemented with 10% fetal bovine serum (Gibco, Middesex, England).Recombinant SEA(wt), SEA(m) and the fusion products C215Fab-SEA(wt) andC215Fab-SEA(m) were obtained as described above. Human recombinant IL-2was from Cetus Corp., USA. Mitomycin C was from Sigma Co., USA. Na₂⁵¹CrO₄ was obtained from Merck, Germany. Phosphate buffered saline (PBS)without magnesium and calcium was received from Imperial, England.

Cells: The human colon carcinoma cell line Colo205 and the B celllymphoma cell line Raji were obtained from American Type Cell CultureCollection (Rockville, Md., USA) (expressing HLA-DR3/w10, -DP7,-DQw1/w2). The EBV-transformed lymphoblastoid B cell line BSM was agenerous gift from Dr van De Griend, Dept of Immunology, Dr Daniel denHoed Cancer Center, Leiden, the Netherlands. The cells were repeatedlytested for mycoplasma contamination with Gen-Probe Mycoplasma T.C. test,Gen-Probe Inc., San Diego, USA.

SEA activated T cell lines were produced by activation of mononuclearcells from peripheral blood. The blood was received as buffy coats fromblood donors at the University Hospital of Lund. The PBMs werestimulated at a concentration of 2×10⁶ cells/ml with mitomycin C treatedSEA coated BSM cells (preincubated with 100 ng/ml SEA) in medium with10% FCS. The T cell lines were restimulated biweekly with 20 U/ml humanrecombinant IL-2 and weekly with mitomycin C treated SEA coated BSMcells. The cell lines were cultivated for 4-12 weeks before being usedin the assay.

The viability of the effector cells, as determined by trypan blueexclusion, exceeded 50%.

Determination of MHC Class II Binding Characteristics of Wild-Type andMutant Sea

Radioiodination Procedure

Appropriate amounts of wild-type or mutant SEA were radiolabeled with 10to 25 mCi Na¹²⁵I using enzymobeads with the lactoperoxidase technique(NEN, Boston, Mass.). The reaction was stopped by quenching with sodiumazide and protein-bound radioactivity was separated from free iodine byfiltration through a PD-10 column (Pharmacia Biotech AB, Sweden) withR10 medium as elution buffer. Conditions were chosen to obtain astoichiometric ratio between iodine-125 and protein of ≦2:1. Theradiochemical purity was verified by size-exclusion chromatography on aTSK SW 3000 HPLC column. The effect of the radioiodination on thebinding activity was only tested for wild-type SEA and found not to beaffected (data not shown).

Direct Binding Assay

Raji cells, 6×10⁴/100 μl, previously cultivated in R10 medium, wereadded to conical polypropylene tubes and incubated (22° C./45 min) intriplicate with 100 μl/tube of serially diluted ¹²⁵I-labeled wild-typeor mutant SEA. The cells were washed with 2 ml 1% (w/v) bovine serumalbumin (BSA) in 10 mM phosphate-buffered saline (PBS), pH 7.4,centrifugated at 300×g for 5 minutes and aspirated. This procedure wasrepeated twice. Finally, the cells were analyzed for cell-boundradioactivity in a gamma counter (Packard Instruments Co, Downers Grove,Ill., USA). The apparent dissociation constant, K_(d), and the number ofbinding sites, N, at saturation were calculated according to Scatchard(Ann. N.Y. Acad. Sci. 51 (1949) 660-72) after subtraction ofnon-specific binding (i.e. binding after incubation with R10 mediumalone.

Inhibition Assay

Inhibition assay (inhibition of ¹²⁵I-labeled wild-type SEA binding bymutant SEAs). These inhibition experiments were carried out as isdescribed for the direct binding assay with slight modifications.Briefly, 50 μl of ¹²⁵I-labeled wild-type SEA was allowed to compete withan excess of unlabeled wild-type or mutant SEA (50 μl/tube) for bindingto 6×10⁴/100 μl Raji cells. A tracer concentration yielding≈40% boundradioactivity in the direct assay was used to obtain maximal sensitivityin the inhibition assay. The displacement capacity of the competitor wasexpressed as the concentration yielding 50% inhibition (IC₅₀) of boundradioactivity. The binding affinity of the mutants relative to wild-typeSEA was calculated using the equation:IC₅₀(SEA(wt)):IC₅₀(SEA(m))

In order to analyze whether the mutants compete for binding to the samesite on Raji cells as wild-type SEA, the binding data obtained with SEAmutants were plotted as a log-logit function and tested for parallelismwith the corresponding data for wild-type SEA.

Inhibition Assay

Inhibition assay (inhibition of the binding of fluorescent-labeledwild-type SEA by unlabeled wild-type SEA and SEA mutants). Raji cells(2.5×10⁵) were incubated with inhibitor (wild-type or mutant SEA; 0-6000nM) diluted in 50 μl CO₂-independent medium (Gibco) supplemented with10% FCS, glutamine and gentamycin at 37° C. for 30 minutes. Fluoresceinconjugated wild-type SEA was added to a final concentration of 30 nM andthe samples were incubated for an additional half hour at 37° C. Thesamples were washed three times with ice cold PBS supplemented with 1%BSA (PBS-BSA) and finally kept in 0.4 ml PBS-BSA on ice until they wereanalyzed. From each sample 10,000 live cells were analyzed for greenfluorescence on a FACStar® (Becton Dickinson) flow cytometer and themean fluorescence value was calculated using the LYSIS II program.

SDCC and SADCC Assays of SEA(wt), SEA(m) and Their Fusion Proteins withC215FAB

SDCC-Assays

The cytotoxicity of SEA(wt), SEA(m) and their fusions with C215Fabagainst MHC class II⁺ Raji cells was analyzed in a standard 4 hour⁵¹Cr³⁺-release assay, using in vitro stimulated SEA specific T celllines as effector cells. Briefly, ⁵¹Cr labeled Raji cells were incubatedat 2.5×10³ cells per 0.2 ml medium (RPMI, 10% FCS) in microtitre wellsat defined effector to target cell ratio in the presence or absence(control) of the additives. Percent specific cytotoxicity was calculatedas 100×([cpm experimental release−cpm background release]/[cpm totalrelease−cpm background release]). The effector to target cell ratio was30:1 for unfused SEAs and 40:1 for fusion proteins.

SADCC Against of Human Colon Cancer Cells

The cytotoxicity of C215Fab-SEA(wt), C215Fab-SEA(m), SEA(wt) and SEAmutants against C²¹⁵⁺ MHC class II⁻ colon carcinoma cells SW 620 wasanalyzed in a standard 4 hour ⁵¹Cr³⁺-release assay, using in vitrostimulated SEA specific T cell lines as effector cells. Briefly,⁵¹Cr³⁺-labeled SW 620 cells were incubated at 2.5×10³ cells per 0.2 mlmedium (RPMI, 10% FCS) in microtitre wells at effector to target cellratio 30:1 in the presence or absence (control) of the additives.Percent specific cytotoxicity was calculated as for SDCC assays.

In Vivo Functional Experiments

Tumor Cells

B16-F1 melanoma cells transfected with a cDNA encoding the human tumorassociated antigen C215 (B16-C215) (Dohlsten et al., Monoclonalantibody-superantigen fusion proteins: Tumor specific agents for T cellbased tumor therapy; Proc. Natl. Acad. Sci. USA, In press, 1994), weregrown as adherent cells to subconfluency. The culture medium consistedof RPMI 1640 (GIBCO, Middlesex, UK) supplemented with 5×10⁻⁵β-mercaptoethanol (Sigma, St Louis, Mo., USA), 2 mM L-glutamine (GIBCO),0.01 M Hepes (Biological Industries, Israel) and 10% fetal calf serum(GIBCO). The cells were detached by a brief incubation in 0.02% EDTA andsuspended in ice cold phosphate buffered saline with 1% syngeneic mouseserum (vehicle) to 4×10⁵ cells/ml.

Animals and Animal Treatment

The mice were 12-19 weeks old C57B1/6 mice transgeneic for a T cellreceptor Vβ3 chain (Dohlsten et al., Immunology 79 (1993) 520-527). Onehundred thousand B16-C215 tumor cells were injected IV in the tail veinin 0.2 ml vehicle. On day 1, 2 and 3, the mice were given IV injectionsof C215Fab-SEA(wt) or C215Fab-SEA(D227A) in 0.2 ml vehicle at dosesindicated in the FIGS. 5 a and 5 b. Control mice were given only vehicleaccording to the same schedule. On day 21 after tumor cell injection,the mice were killed by cervical dislocation, the lungs removed, fixedin Bouin's solution and the number of lung metastases counted.

Results

“Alanine Scanning” of Staphylococcal Enterotoxin A

Initially the structure of SEA was unknown and only speculations couldbe done about what side chains were surface accessible. Therefore, themajority of the mutants were chosen from alignments of homologoussuperantigens (Marrack and Kappler, Science 248 (1990) 705-711).Conserved (mainly polar) residues were chosen on the rational that someof these superantigens are expected to bind to HLA-DR in a conservedfashion (Chitagumpala et al., J. Immunol. 147 (1991) 3876-3881). Alaninereplacements were used according to published strategies (Cunnninghamand Wells, Science 244 (1988) 1081-1085). During the course of this workthe available information increased: i) it was shown that a Zn²⁺ ion isimportant for the interaction between SEA and MHC class II (HLA-DR)(Fraser et al., Proc. Natl. Acad. Sci. USA 89 (1991) 5507-5511), ii) amutational analysis of staphylococcal enterotoxin B (SEB) was presented(Kappler et al., J. Exp. Med. 175 (1992) 387-396), and iii) thestructure of SEB was presented (Swaminathan et al., Nature 359 (1992)801-806).

Our first mutant showing a severely reduced affinity for HLA DR, D227A,was found to coordinate the Zn²⁺ ion very poorly (data not shown).Assuming a common fold for SEA and SEB, the new data suggested two MHCclass II binding regions; one involving the Zn²⁺ ion and onecorresponding to the site defined in SEB. A second set of mutations weremade on these assumptions. This second set of mutants were expressed inthe form of SEA carrying a glycine added at the amino terminus. Firstthe extension was shown to have no effects on the binding properties ofwild-type SEA (next section).

Most of the mutants were expressed and secreted by E. coli in afunctional form as judged by analysis of the binding of monoclonalantibodies (Table I). Very low amounts were obtained of the mutantsE154A/D156A and R160A. Consequently these were excluded from the study.The mutants having an Ala substitution in residues 128, 187, 225 or 227were not recognized by the monoclonal antibody 1E. The latter twomutants showed a reduced level of expression (more pronounced at 34° C.than at 24° C.) and migrated faster during SDS-PAGE, under denaturingbut not reducing conditions (all other mutants migrated as wild-typeSEA, data not shown). As judged by CD spectra analysis the structure ofD227A could differ slightly from native SEA (FIG. 2), but the stabilitywas very close to wild-type SEA (measured as resistance towardsguanidine hydrochloride denaturation). The calculated ΔΔG between themutant and native SEA (SEA(wt)) was 0.16 kcal/mol and is only about 4%of the ΔG° values (data not shown). Overall the signals in the CDanalysis were low, as expected from a mostly β-sheet structure. It wasrecently reported that His 225 coordinates Zn²⁺ (unpublished data inFraser et al (Proc. Natl. Acad. Sci. USA 89 (1991) 5507-5511). Since Asp227 is involved in Zn²⁺ coordination (above) and presumably located inthe same β-sheet as His 225 this suggests that these two residuesconstitutes the zinc-binding nucleus found in zinc-coordinating proteins(Vallee and Auld, Biochemistry 29 (1990) 5647-5659).

Binding to MHC class II and T Cell Receptor

The MHC class II affinity was calculated from the amounts needed tocompete with fluorescein-labeled wild-type SEA for Raji cell exposinglarge amounts of MHC class II. The displacement capacity of a mutant wascalculated from the concentration yielding 50% inhibition (IC₅₀) ofbound fluorescence compared with the concentration needed with wild-typeSEA as the competitor. For wild-type SEA and for some mutants, theresult from this analysis was compared with the result from an analysiswhere ¹²⁵I labeled wild-type SEA was used as the tracer. As may be seenin Table II, the values obtained from these two inhibition analysescorrelate well.

For six selected mutants the binding to MHC class II was measureddirectly using ¹²⁵I labeled mutant SEA (Table II). With the mutant H50Athe values obtained from the direct binding assay and the inhibitionassays correlated well but with the mutant F47A a large discrepancy wasfound: the direct binding indicated only 7 times weaker binding thanwild-type SEA but both competition analyses demonstrated around 70 timesreduced binding. The data from two of the other mutants indicated twoseparate binding interactions. For the mutants H225A and D227A theaffinity was below the detection limit also in this analysis.

We previously showed that fusion proteins composed of the Fab fragmentof a carcinoma reactive antibody and SEA could be used to directcytotoxic T cells to specifically lyse cancer cells, while theinteraction between SEA and the T cell receptor (TCR) was too weak to bedetected by itself (Dohlsten et al., Proc. Natl. Acad. Sci. USA, inpress). Thus, in contrast to analyses involving the isolatedsuperantigen the Fab fusion context enables a functional assay for theinteraction between SEA and the TCR, independent of the MHC class IIbinding. Consequently, the efficiency of the different conjugates todirect T cells to lyse cells recognized by the Fab moiety was monitoredin a chromium release assay. This analysis confirmed that the mutationsshown to affect the MHC class II binding did not affect the TCR binding(Table II).

Biological Effects of the Mutations

The proliferative effect was measured as the ability to stimulateperipheral lymphocytes to divide. All three mutants that competes verypoorly for MHC class II induced little or no proliferation and theintermediate mutant H187A displayed some proliferative capacity, whereasthe other investigated mutants were indistinguishable from the wild-type(table III). Harris et al (Infect. Immun. 61 (1993) 3175-3183) recentlyreported a similar severe reduction in T cell stimulatory activity forthe SEA mutants F47G and L48G. Clearly a strong reduction in any of thetwo suggested binding regions results in a severe effect on the abilityto induce proliferation. This suggests that SEA cross-links twomolecules of MHC class II leading to dimerization of the TCR and thatthis is needed to yield a signal transduction.

In contrast the efficiency of the different mutants in directing invitro stimulated SEA T cells to lyse MHC class II bearing target cellsshows correlation with the binding affinity, rather than to the abilityto compete (Table III). For example, the efficiency of F47A and D227Aare only reduced 2.5 times and 300 times, respectively. Thus, here noinherent requirement for divalency too is obvious. The increase inmultivalency resulting from the significantly larger number of TCRs onthe surface of activated T cells might partially shield the effect of alower avidity in the SEA/MHC class II interaction. That dimerization isnot needed to direct T cell cytotoxicity has previously beendemonstrated by the use of carcinoma specific bifunctional antibodiescontaining one anti-CD3 moiety and one anti-carcinoma moiety (Renner etal., Science 264 (1994) 833-35).

In Vivo Functional Experiments

The results are represented in FIGS. 6 a and 6 b. Treatment of mice withC215Fab-SEA(wt) and C215Fab-SEA(D227A) were both highly effective inreducing the number of lung metastases of B16-C215 melanoma cells. Thetherapeutic effect was essentially identical for the two variants of thetargeted superantigens. Treatment with C215Fab-SEA(wt) resulted in 70%lethality at doses of 5 μg/injection. In contrast, no mice died when thesame dose of C215Fab-SEA(D227A) were used. Taken together, SEA(D227A) isan example of a mutant with reduced toxicity and retained therapeuticefficiency when incorporated in a Fab-SEA fusion protein.

Discussion

The structure of the complex between SEB and HLA-DR was recentlyreported (Jardetzky et al., Nature 368 (1994) 711-718). Most of the SEBresidues identified to be involved in this interaction are conserved inSEA. Our data on mutant D227A indicates a weak affinity for theinteraction between this site of SEA (the amino proximal site) and theMHC class II, having a K_(d) value higher than 8 μM. The K_(d) for theinteraction between SEB and HLA-DR was recently reported to be 1.7 μM(Seth et al., Nature 369 (1994) 324-27). The different interactionsbetween SEB, TCR and HLA-DR were investigated and it was shown that thecomplex between SEB and HLA-DR was not stably maintained in the absenceof TCR. Plasmon resonance experiments indicated that this was because ofa very fast off-rate. The avidity effects obtained if SEA cross-linkstwo molecules of MHC class II followed by a subsequent dimerization ofthe TCR could explain how SEA may induce proliferative effects atconcentrations well below the K_(d). Assuming that the mutation F47Areduces the affinity of the amino proximal site below significance, theK_(d) of the Zn²⁺ site is around 95 nM. This hypothesis was recentlystrengthened by the observation that the mutants F47R, F47R/H50A andF47R/L48A/H50D show identical affinity for MHC class II as F47A(unpublished).

Based on the-SEB structure (Kappler et al., J. Exp. Med. 175 (1992)387-396) and on homology alignments (Marrack and Kappler, Science 248(1990) 705-711), it is strongly suggested that His225 and Asp227 arelocated in the same β-sheet and thus the side chains could be proximal.Thus, most likely these two residues constitute the zinc-binding nucleusfound in zinc-coordinating proteins (Vallee and Auld, Biochemistry 29(1990) 5647-5659). Similarly to these mutants, the mutants with areplacement at residue 128 or 187 are also recognized by all monoclonalsexcept 1E. Fraser et al (Proc. Natl. Acad. Sci. USA 89 (1991) 5507-5511)showed that Zn²⁺ is bound to SEA and is needed for a high affinityinteraction with MHC class II. The affinity for zinc was not affected bythe addition of HLA-DR. Based on this observation and the high affinityfor Zn²⁺ (K_(d) of around 1 μM) a coordination exclusively provided bySEA and involving 4 fold coordination was suggested. Our data indicatesan involvement of the four residues N128, H187, H225 and D227. Thefunction of the former two residues is not yet clear; instead ofproviding a ligand N128 could help in the deprotonation of D227. Oneargument for this is that the effect of replacing D227 is more severethat when replacing H225.

It was previously reported that there is a lack of correlation betweenthe affinity of different superantigens for the MHC class II and thecapacity to stimulate T cells to proliferate (Chintagumpala et al., J.Immunol. 147 (1991) 3876-3881). These results might partly be explainedby different affinities of the superantigens towards different TCR Vβ-chains. Here we have observed the same lack of correlation but incontrast to separate superantigens the mutants display identical TCRaffinity as shown in the Fab-SEA context (measured as SADCC). The mostlikely explanation for the lack of correlation is that two bindingregions identified in this analysis represent two separate binding sitesthat yields not only a co-operative binding, but which results in thecross-linking of two molecules of MHC class II, which in turn yieldsdimerization of two molecules of the T cell receptor. This would implythat the affinity of both sites are important to obtain theproliferative effect. A high avidity results from the interactionswithin a hexameric complex involving two molecules of SEA, TCR and MHCclass II. Thus the strong affinity/avidity of SEA towards MHC class IIenables SEA interaction with the TCR despite a low direct affinity.

Other Biospecific Affinity Counterparts

A fusion protein of SEA(D227A) and an IgG-binding domain ofstaphylococcal protein A has been produced by recombinant technology andexpressed in E. coli. This reagent has successfully been used to targetT-lymphocytes to Mot 4 and CCRF-CEM cells (obtained from ATCC) that areCD7 and CD38 positive but HLA-DP, -DQ and -DR negative. The Mot 4 andCCRF-CEM cells were preincubated with anti-CD7 or anti-CD38 mousemonoclonals (Dianova, Hamburg, Germany). In order to enhance bindingbetween the mouse monoclonals and the IgG-binding part of the fusionprotein rabbit anti-mouse Ig antibody was also added.

In comparison with protein A-SEA(wt), protein A-SEA(D227A) had adecreased ability to bind to Daudi cells expressing MHC class IIantigen. TABLE I Confirmation of mutant structural integrity. Thebinding of six monoclonal antibodies was monitored. Monoclonal antibodyMutation 1A 2A 3A 1E 4E EC-A1 Wild-type + + + + + +D11A/K14A + + + + + + D45A + + + + + + F47A + + + + + + H50A (+) +(+) + + + K55A + + + + + + H114A + + + + + + K123A/D132G + + + + + +N128A + + + − + + K147A/K148A + + + + − + E154A/D156A ND ND ND + ND NDR160A ND ND ND + ND ND H187A + + + − + + E191A/N195A + + + + + +D197A + + + + + + H225A + + + − + + D227A + + + − + +Footnotes:A plus sign indicates binding, parenthesis indicate 50 to 90% bindingcompared with wild-type SEA. ND means not determined.

TABLE II Binding of SEA mutants to the MHC class II and the T cellreceptor. The latter was monitored as the ability to direct activatedcytotoxic T-cells specifically to lyse carcinoma cells using Fab-SEAfusions of the different mutants (SADCC). IC₅₀(nM) IC₅₀(nM) K_(d)(nM)SADCC(% of Mutation SEA-FITC¹ 125_(I-SEA) ¹ 125_(I labeled) ¹ wild-type¹wild-type 50 38 13  100² Gly-SEA 50 ND ND  100² D11A/K14A 50 ND ND NDD45A 53 ND ND ND F47A 3150 2943 95 100 H50A 150 132 32 100 K55A 44 ND NDND H114A 48 ND ND ND K123A/D132G 188 75 12/237 100 N128A 1150 ND 2.9/76 100 K147A/K148A 58 ND ND ND H187A 1030 602 97 100 E191A/N195A 51 ND NDND D197A 78 ND ND ND H225A >9000 9600 ND ND D227A >9000 >10000 >8000 100Footnotes:¹ND means not determined.²In the Fab-SEA context the spacer between C_(H)i and SEA ends with aGly.

TABLE III Biological effects of the mutations. The ability to stimulateresting T cells to proliferate and the ability to direct cytotoxic cellsto lyse MHC class II exposing target cells were monitored (SDCC =Superantigen Dependent mediated Cellular Cytotoxicity). ProliferationSDCC Mutation % EC₅₀ (relative) wild-type 100 1 Gly-SEA ND 1 D11A/K14AND 0.8 D45A 50 1.3 F47A <0.2 2.5 H50A 20 1.4 K55A 100 1.3 H114A ND 1K123A/D132G 40 2.1 N128A 40 1.2 K147A/K148A ND 0.7 E154A/D156A ND NDR160A ND ND H187A 15 4 E191A/N195A 100 1.1 D197A ND 1.3 H225A <0.2 3 ×10² D227A <0.01 3 × 10²Footnotes:ND means not determined.

1-13. (canceled)
 14. A method for the lysis of cells associated with adisease condition in a mammal, which condition is associated with thepresence of cells expressing a specific cell surface structure,comprising the step of: administering to the mammal a therapeuticallyeffective amount of a comprising a bacterial superantigen and abiospecific affinity counterpart, wherein (1) the biospecific affinitycounterpart is capable of binding to a specific cell surface structure,and (2) the superantigen comprises a peptide that: i. contains an aminoacid sequence that is derived from Staphylococcal enterotoxin A, whereinsaid peptide has the ability to bind to a Vβ chain of a T cell receptor,and ii. has been modified at an amino acid position 47, 128, 187, 225 or227, in order to have reduced ability to bind to MHC class II antigens.15. The method of claim 14, wherein said amino acid modification is asubstitution to alanine.
 16. The method of claim 14, wherein said aminoacid modification is a substitution that is not a conserved substitutionof the amino acid at that position in Staphylococcal enterotoxin A. 17.The method of claim 14, wherein the specific cell surface structure is acancer specific epitope.
 18. The method of claim 17, wherein thespecific cell surface structure is the C242 epitope.
 19. The method ofclaim 14, wherein the biospecific affinity counterpart is an antibody oran antigen-binding fragment of an antibody.
 20. The method of claim 19,wherein the biospecific affinity counterpart is an antigen-bindingfragment of an antibody.
 21. The method of claim 20, wherein thefragment of an antibody is selected from the group consisting of Fab,F(ab)₂, Fv, and single chain antibody.
 22. The method of claim 19,wherein the biospecific affinity counterpart is monoclonal antibodyC242.
 23. The method of claim 19, wherein the biospecific affinitycounterpart is monoclonal antibody C215.
 24. The method of claim 14,wherein the modification is at amino acid position
 47. 25. The method ofclaim 14, wherein the modification is at amino acid position
 128. 26.The method of claim 14, wherein the modification is at amino acidposition
 187. 27. The method of claim 14, wherein the modification is atamino acid position
 225. 28. The method of claim 14, wherein themodification is at amino acid position
 227. 29. The method of claim 14,wherein the modification is a substitution to serine at amino acidposition
 227. 30. The method of claim 14, wherein the modification is atamino acid positions 47, 128, 187, 225 and
 227. 31. The method of claim15, wherein the modification is at amino acid positions
 47. 32. Themethod of claim 15, wherein the modification is at amino acid position128.
 33. The method of claim 15, wherein the modification is at aminoacid position
 187. 34. The method of claim 15, wherein the modificationis at amino acid position
 225. 35. The method of claim 15, wherein themodification is at amino acid position
 227. 36. The method of claim 15,wherein the modification is at amino acid positions 47, 128, 187, 225and
 227. 37. The method of claim 16, wherein the modification is atamino acid position
 47. 38. The method of claim 16, wherein themodification is at amino acid position
 128. 39. The method of claim 16,wherein the modification is at amino acid position
 187. 40. The methodof claim 16, wherein the modification is at amino acid position
 225. 41.The method of claim 16, wherein the modification is at amino acidposition
 227. 42. The method of claim 16, wherein the modification is atamino acid positions 47, 128, 187, 225 and
 227. 43. A method for thelysis of cells associated with a disease condition in a mammal, whichcondition is associated with the presence of cells expressing a specificcell surface structure, comprising the step of: administering to themammal a therapeutically effective amount of a pharmaceuticalcomposition comprising: (A) a conjugate comprising a bacterialsuperantigen and a biospecific affinity counterpart, wherein (1) thebiospecific affinity counterpart is capable of binding to a specificcell surface structure, and (2) the superantigen comprises a peptidethat: i. contains an amino acid sequence that is derived fromStaphylococcal enterotoxin A, wherein said peptide has the ability tobind to a Vβ chain of a T cell receptor, and ii. has been modified at anamino acid position 47, 128, 187, 225 or 227, in order to have reducedability to bind to MHC class II antigens; and (B) a pharmaceuticallyacceptable vehicle.
 44. The method of claim 43, wherein said amino acidmodification is a substitution to alanine.
 45. The method of claim 43,wherein said amino acid modification is a substitution that is not aconserved substitution of the amino acid at that position inStaphylococcal enterotoxin A.
 46. The method of claim 43, wherein thespecific cell surface structure is a cancer specific epitope.
 47. Themethod of claim 46, wherein the specific cell surface structure is theC242 epitope.
 48. The method of claim 43, wherein the biospecificaffinity counterpart is an antibody or an antigen-binding fragment of anantibody.
 49. The method of claim 48, wherein the biospecific affinitycounterpart is an antigen-binding fragment of an antibody.
 50. Themethod of claim 49, wherein the fragment of an antibody is selected fromthe group consisting of Fab, F(ab)₂, Fv, and single chain antibody. 51.The method of claim 48, wherein the biospecific affinity counterpart ismonoclonal antibody C242.
 52. The method of claim 48, wherein thebiospecific affinity counterpart is monoclonal antibody C215.
 53. Themethod of claim 43, wherein the modification is at amino acid position47.
 54. The method of claim 43, wherein the modification is at aminoacid position
 128. 55. The method of claim 43, wherein the modificationis at amino acid position
 187. 56. The method of claim 43, wherein themodification is at amino acid position
 225. 57. The method of claim 43,wherein the modification is at amino acid position
 227. 58. The methodof claim 43, wherein the modification is a substitution to serine atamino acid position
 227. 59. The method of claim 43, wherein themodification is at amino acid positions 47, 128, 187, 225 and
 227. 60.The method of claim 44, wherein the modification is at amino acidposition
 47. 61. The method of claim 44, wherein the modification is atamino acid position
 128. 62. The method of claim 44, wherein themodification is at amino acid position
 187. 63. The method of claim 44,wherein the modification is at amino acid position
 225. 64. The methodof claim 44, wherein the modification is at amino acid position
 227. 65.The method of claim 44, wherein the modification is at amino acidpositions 47, 128, 187, 225 and
 227. 66. The method of claim 45, whereinthe modification is at amino acid position
 47. 67. The method of claim45, wherein the modification is at amino acid position
 128. 68. Themethod of claim 45, wherein the modification is at amino acid position187.
 69. The method of claim 45, wherein the modification is at aminoacid position
 225. 70. The method of claim 45, wherein the modificationis at amino acid position
 227. 71. The conjugate of claim 45, whereinthe modification is at amino acid positions 47, 128, 187, 225 and 227.